Analyzing Messenger RNA and Micro RNA in the Same Reaction Mixture

ABSTRACT

The present teachings provide methods, compositions, and kits for performing primer extension reactions on at least two target polynucleotides in the same reaction mixture. In some embodiments, a reverse transcription reaction is performed on a first target polynucleotide with a hot start primer comprising a self-complementary stem and a loop, and extension products form at high temperatures but extension products form less so at low temperatures since the self-complementary stem of the hot start primer prevents hybridization of the target specific region to the target. However, non-hot start primers with free target specific regions can hybridize to their corresponding targets at the low temperature and extension can happen at the low temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims a priority benefit under 35 U.S.C. §119(e) fromU.S. Application No. 60/699,930, filed Jul. 15, 2005, the contents ofwhich is incorporated herein by reference.

FIELD

The present teachings relate to primer-extension mediated methods ofsynthesizing nucleic acids.

INTRODUCTION

The integrity of primer-mediated methods of synthesizing nucleic acidscan be compromised by non-specific hybridization of primer toinappropriate target polynucleotides. The analysis of nucleic acids isbenefited by approaches that minimize the generation of mis-extensionproducts. For example, ‘hot-start’ approaches have been employed in PCR,where inhibition of polymerase activity has been achieved. For example,U.S. Pat. No. 5,338,671 describes the use of antibodies specific for athermostable DNA polymerase to inhibit the DNA polymerase activity atlow temperatures. Chemical treatment with citraconic anhydride isanother way hot-start PCR has been achieved (see U.S. Pat. No. 5,773,258and U.S. Pat. No. 5,677,152). Hot-start methods which use a heat labilematerial, such as wax, to separate or sequester reaction components aredescribed in U.S. Pat. No. 5,411,876. The application of such hot-startapproaches to reverse transcription have proven challenging. Forexample, many reverse transcriptases are not heat-stabile.

Analysis of expressed nucleic acids can be difficult in small andlimited samples. Approaches that multiplex nucleic acid analyses are ofgrowing importance in the biomedical research community.

SUMMARY

In some embodiments, the present teachings provide a method of forming amessenger RNA (mRNA) primer extension product and a micro RNA primerextension product in the same reaction mixture comprising; forming areverse transcription reaction mixture comprising a hot start mRNAprimer, a micro RNA primer, a target mRNA, a target micro RNA, and areverse transcribing enzyme, wherein the hot start mRNA primer comprisesa blunt-ended self-complementary stem and a loop, wherein theblunt-ended self-complementary stem comprises a target specific regionand a quencher region; hybridizing the micro RNA primer to the targetmicro RNA at a low temperature; extending the micro RNA primer at thelow temperature to form a micro RNA extension product, wherein theself-complementary stem of the mRNA primer is substantiallynon-denatured; raising the temperature to a high temperature, whereinthe self-complementary stem of the hot start mRNA primer issubstantially denatured; hybridizing the target specific region of thehot start mRNA primer to the target mRNA at the high temperature;extending the hot start mRNA primer at the high temperature to form amRNA extension product; and, forming the mRNA primer extension productand the micro RNA primer extension product in the same reaction mixture.

In some embodiments, the present teachings provide a method of forming afirst primer extension product and a second primer extension product inthe same reaction mixture comprising; forming a primer extensionreaction mixture comprising a first primer, a hot start second primer, afirst target, a second target, and a primer extending enzyme, whereinthe hot start second primer comprises a blunt-ended self-complementarystem and a loop, wherein the blunt-ended self-complementary stemcomprises a target specific region and a quencher region; hybridizingthe first primer to the first target at a low temperature; extending thefirst primer at the low temperature to form a first primer extensionproduct, wherein the self-complementary stem of the hot start secondprimer is substantially non-denatured; raising the temperature to a hightemperature, wherein the self-complementary stem of the hot start secondprimer is substantially denatured; hybridizing the target specificregion of the hot start second primer to the second target at the hightemperature; extending the hot start second primer at the hightemperature to form a second target extension product; and, forming thefirst primer extension product and the second primer extension productin the same reaction mixture.

In some embodiments, the present teachings provide a reaction mixturecomprising a hot start mRNA primer, a micro RNA primer, a target mRNA, atarget micro RNA, and a reverse transcribing enzyme, wherein the hotstart mRNA primer comprises a blunt-ended self-complementary stem and aloop, wherein the blunt-ended self-complementary stem comprises a targetspecific region and a quencher region, wherein the self-complementarystem of the hot start mRNA primer is substantially non-denatured at alow temperature, and wherein the self-complementary stem of the hotstart mRNA primer is substantially denatured at a high temperature.

In some embodiments, the present teachings provide a reaction mixturecomprising a hot start mRNA primer, a micro RNA primer, a target mRNA, atarget micro RNA and a reverse transcribing enzyme, wherein the hotstart mRNA primer comprises a blunt-ended self-complementary stem and aloop, wherein the blunt-ended self-complementary stem comprises a targetspecific region and a quencher region, wherein the self-complementarystem of the hot start mRNA primer is non-denatured at a low temperature,and wherein the self-complementary stem of the hot start mRNA primer isdenatured at a high temperature

In some embodiments, the present teachings provide a kit for forming amessenger RNA (mRNA) primer extension product and a micro RNA (miRNA)primer extension product in the same reaction mixture comprising; a hotstart mRNA primer, a micro RNA primer, and optionally a reversetranscribing enzyme, wherein the hot start mRNA primer comprises ablunt-ended self-complementary stem and a loop, wherein the blunt-endedself-complementary stem comprises a target specific region and aquencher region, wherein the self-complementary stem is substantiallynon-denatured at a low temperature, wherein the low temperature is lessthan 27 C, and wherein the self-complementary stem is substantiallydenatured at a high temperature, wherein the high temperature is between35 C-60 C.

These and other features of the present teachings are set forth herein.

DRAWINGS

FIG. 1 depicts one illustrative embodiment according to the presentteachings.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not intended to limit the scope of the current teachings. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. For example, “a forward primer” meansthat more than one forward primer can be present; for example, one ormore copies of a particular forward primer species, as well as one ormore different forward primer species. Also, the use of “comprise”,“contain”, and “include”, or modifications of those root words, forexample but not limited to, “comprises”, “contained”, and “including”,are not intended to be limiting. The term and/or means that the termsbefore and after can be taken together or separately. For illustrationpurposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or“X and Y”.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. All literature and similar materials cited in this application,including, patents, patent applications, articles, books, treatises, andinternet web pages are expressly incorporated by reference in theirentirety for any purpose. In the event that one or more of theincorporated literature and similar defines or uses a term in such a waythat it contradicts that term's definition in this application, thisapplication controls. While the present teachings are described inconjunction with various embodiments, it is not intended that thepresent teachings be limited to such embodiments. On the contrary, thepresent teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

SOME DEFINITIONS

As used herein, the term “hot start primer” refers to a primercomprising a self-complementary stem and a loop, wherein theself-complementary stem comprises a target specific region and aquencher region. At low temperatures, the target-specific region ishybridized to the quencher region. At high temperatures, thetarget-specific region is not hybridized to the quencher region, and canhybridize to the corresponding target polynucleotide, thereby allowingfor a hot-start extension reaction. In some embodiments, theself-complementary stem is blunt-ended, such that there is not anucleotide overlap at the 5′ or 3′ end of the self-complementary stem.In some embodiments, the mRNA primer comprises a nearly blunt-endedself-complementary stem, such as for example a single 3′ nucleotideoverhang. Generally, such 3′ overhangs will be of minimal length toavoid undesired priming on targets prior to the melting of theself-complementary stem region by the high temperature. Overhangs on the5′ end are generally more tolerable, since extension does not proceedfrom the 5′ of a sequence.

As used herein, the term “pre-amplifying” refers to a process wherein amultiplexed PCR is performed, followed by a plurality of lower-plexdecoding PCRs. Typically the primers employed in the multiplexed PCRcorrespond to the primers employed in the plurality of lower-plexdecoding PCRs. Illustrative teachings of such approaches can be found inWO2004/051218 to Andersen and Ruff, U.S. Pat. No. 6,605,451 to Xtrana,and U.S. Non-Provisional application Ser. No. 11/090,830 to Andersen etal., and U.S. Non-Provisional application Ser. No. 11/090,468 to Lao etal.,

As used herein, the term “denaturing” refers to the melting of twocomplementary nucleic acid strands, and is typically achieved byelevating the temperature. In some embodiments, denaturing can beachieved by the addition of base (e.g. —NaOH) or other approaches fordissociating nucleic acids that are familiar to one of ordinary skill inthe art of molecular biology.

As used herein, the term “complementary” refers to nucleic acidsequences that are capable of forming Watson-Crick base-pairs. Forexample, a self-complementary primer comprises a self-complementary stemwhich is capable of forming Watson-Crick base-pairs with itself at a lowtemperature. When at the low temperature, the strands of such aself-complementary stem are said to be hybridized to one another. Whenat a high temperature, the strands of such a self-complementary stem arenot hybridized to each other, and the target specific region of theself-complementary stem can be hybridized with a target. In thisapplication, a statement that one sequence is complementary to anothersequence encompasses situations in which the two sequences have slightmismatches. Here, the term “sequence” encompasses, but is not limitedto, nucleic acid sequences, polynucleotides, oligonucleotides, probes,primers, primer-specific regions, and target-specific regions. Despitethe mismatches, the two sequences should selectively hybridize to oneanother under appropriate conditions.

As used herein, the term “blunt-ended self-complementary stem” refers toan aspect of a hot start primer that is double-stranded and hybridizesto itself at a low temperature. In some embodiments, the stem isblunt-ended, in that there are not any overlapping, non-hybridizednucleotides, at the end of the hot start primer when the rest of thenucleotides in the stem are hybridized in base-pairs. In someembodiments, the stem has one, or two, or as many as three overlappingnucleotides, and possibly more, but for the purposes of the presentteachings such a molecule can still be considered “blunt-ended.” Thus,small overlaps, which are not expected to prime extension of a targetwhen the rest of the stem is double-stranded, are within the scope ofblunt-ended self-complementary stems of the hot start primers providedby the present teachings.

As used herein, the term “target polynucleotide” refers to apolynucleotide sequence that is sought to be reverse transcribed. Thetarget polynucleotide can be obtained from any source, and can compriseany number of different compositional components. For example, thetarget can be nucleic acid (e.g. DNA or RNA), transfer RNA, siRNA, andcan comprise nucleic acid analogs or other nucleic acid mimic, thoughtypically the target will be messenger RNA (mRNA) and/or micro RNA(miRNA). The target can be methylated, non-methylated, or both. Thetarget can be bisulfite-treated and non-methylated cytosines convertedto uracil. Further, it will be appreciated that “target polynucleotide”can refer to the target polynucleotide itself, as well as surrogatesthereof, for example amplification products, and native sequences. Insome embodiments, the target polynucleotide is a short DNA moleculederived from a degraded source, such as can be found in for example butnot limited to forensics samples (see for example Butler, 2001, ForensicDNA Typing: Biology and Technology Behind STR Markers. The targetpolynucleotides of the present teachings can be derived from any of anumber of sources, including without limitation, viruses, prokaryotes,eukaryotes, for example but not limited to plants, fungi, and animals.These sources may include, but are not limited to, whole blood, a tissuebiopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen,biowarfare agents, anal secretions, vaginal secretions, perspiration,saliva, buccal swabs, various environmental samples (for example,agricultural, water, and soil), research samples generally, purifiedsamples generally, cultured cells, and lysed cells. It will beappreciated that target polynucleotides can be isolated from samplesusing any of a variety of procedures known in the art, for example theApplied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABIPrism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat.No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will beappreciated that target polynucleotides can be cut or sheared prior toanalysis, including the use of such procedures as mechanical force,sonication, restriction endonuclease cleavage, or any method known inthe art. In general, the target polynucleotides of the present teachingswill be single stranded, though in some embodiments the targetpolynucleotide can be double stranded, and a single strand can resultfrom denaturation.

As used herein, the term “nucleotide” refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR₂ or halogen groups, where each Ris, independently H, C₁-C₆ alkyl or C₅-C₁₄ aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-a-anomeric nucleotides, V-a-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352, andWO 99/14226). Exemplary LNA sugar analogs within a polynucleotideinclude, but are not limited to, the structures:

where B is any nucleotide base.

Modifications at the 2′- or 3′-position of ribose include, but are notlimited to, hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy,butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino,alkylamino, fluoro, chloro and bromo. Nucleotides include, but are notlimited to, the natural D optical isomer, as well as the L opticalisomer forms (see, e.g., Garbesi (1993) Nucl. Acids Res. 21:4159-65;Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993) NucleicAcids Symposium Ser. No. 29:69-70). When the nucleotide base is purine,e.g. A or G, the ribose sugar is attached to the N⁹-position of thenucleotide base. When the nucleotide base is pyrimidine, e.g. C, T or U,the pentose sugar is attached to the N′-position of the nucleotide base,except for pseudouridines, in which the pentose sugar is attached to theC5 position of the uracil nucleotide base (see, e.g., Kornberg andBaker, (1992) DNA Replication, 2^(nd) Ed., Freeman, San Francisco,Calif.).

One or more of the pentose carbons of a nucleotide may be substitutedwith a phosphate ester having the formula:

where α is an integer from 0 to 4. In certain embodiments, α is 2 andthe phosphate ester is attached to the 3′- or 5′-carbon of the pentose.In certain embodiments, the nucleotides are those in which thenucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analogthereof. “Nucleotide 5′-triphosphate” refers to a nucleotide with atriphosphate ester group at the 5′ position, and are sometimes denotedas “NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. The triphosphate ester group may includesulfur substitutions for the various oxygens, e.g. α-thio-nucleotide5′-triphosphates. For a review of nucleotide chemistry, see: Shabarova,Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH,New York, 1994.

FIG. 1 depicts a method of forming a messenger RNA (mRNA) primerextension product and a micro RNA primer extension product in the samereaction mixture according to some embodiments of the present teachings.

In FIG. 1 (A), a reaction mixture is shown comprising a micro RNA target(1) a mRNA target (2), a micro RNA primer (3), and a mRNA primer (4), ata low temperature, for example 25 C room temperature. The mRNA primer(4) is a hot start primer, and comprises a loop (5) and aself-complementary stem (6), wherein the self complementary stemcomprises a target specific region (7) and a quencher region (8). The 3′end of the target specific region (7) of the mRNA primer (4) isdepicted. The poly-A tail at the 3′ end of the mRNA target (2) isdepicted as a series of A's.

In FIG. 1 (B), the micro RNA primer (3) hybridizes to the micro RNAtarget (1) and is extended (bold arrow), while the hot start mRNA primer(4) remains substantially non-denatured, and cannot hybridize to itscorresponding mRNA target at the low temperature.

In FIG. 1 (C), the reaction mixture is heated to a high temperature, forexample 42 C, and the hot start mRNA primer (4) is denatured, thusliberating the target-specific region (7) from the quencher region (8),with the loop (5) shown disposed therein. Also depicted in FIG. 1 (C) isthe micro RNA extension product (9) and the target mRNA (2)

In FIG. 1 (D), the target specific region (7) of the hot start mRNAprimer (4) is shown hybridized to the mRNA target (2) and is extended(bold arrow). The quencher region (8) and the loop (5) of the hot startmRNA primer, which are single-stranded, are also depicted.

In some embodiments, the architecture of the hot start primer can differfrom that depicted in FIG. 1. For example, the loop (6) of the hot startprimer shown in FIG. 1 can itself form part of the target-specificregion of the hot start primer. It will be appreciated that one of skillin the art would be able to make these and other minor modifications inthe design of the hot start primer provided by the present teachings,and still be within their scope.

One of skill in the art will appreciate that the methods, compositions,and kits of the present teachings can be useful in a variety of contextsin which primer-extension on different target polynucleotides is desiredto be performed in the same reaction mixture. In some embodiments of thepresent teachings, messenger RNA and micro RNA can be reversetranscribed using the methods of the present teachings. In someembodiments, the messenger RNA and micro RNA can be found in a smallsample, for example a small sample derived from laser capturemicrodissection.

Certain methods of optimizing reverse transcription and amplificationreactions are known to those skilled in the art. For example, it isknown that PCR may be optimized by altering times and temperatures forannealing, polymerization, and denaturing, as well as changing thebuffers, salts, and other reagents in the reaction composition.Optimization may also be affected by the design of the amplificationprimers used. For example, the length of the primers, as well as theG-C:A-T ratio may alter the efficiency of primer annealing, thusaltering the amplification reaction. Descriptions of amplificationoptimization can be found in, among other places, James G. Wetmur,“Nucleic Acid Hybrids, Formation and Structure,” in Molecular Biologyand Biotechnology, pp. 605-8, (Robert A. Meyers ed., 1995); McPherson,particularly in Chapter 4; Rapley; and Protocols & Applications Guide,rev. 9/04, Promega.

In some embodiments, the present teachings contemplate single-tubeRT-PCR approaches, and discussed for example in Mohamed et al., (2004)Journal of Clinical Virology, 30:150-156. In some embodiments, thereverse transcription products of the present teachings can be amplifiedin a multiplexed pre-amplifying PCR followed by a plurality oflower-plex decoding PCRs, as described for example in WO2004/051218 toAndersen and Ruff, U.S. Pat. No. 6,605,451 to Xtrana, and U.S.Non-Provisional application Ser. No. 11/090,830 to Andersen et al., andU.S. Non-Provisional application Ser. No. 11/090,468 to Lao et al. Thusin some embodiments, the present teachings provide a method ofquantifying a messenger RNA (mRNA) and a micro RNA from a small samplecomprising; forming a reverse transcription reaction mixture comprisinga hot start mRNA primer, a micro RNA primer, a target mRNA, a targetmicro RNA, and a reverse transcribing enzyme, wherein the hot start mRNAprimer comprises a blunt-ended self-complementary stem and a loop,wherein the blunt-ended self-complementary stem comprises a targetspecific region and a quencher region; hybridizing the micro RNA primerto the target micro RNA at a low temperature; extending the micro RNAprimer at the low temperature to form a micro RNA extension product,wherein the self-complementary stem of the hot start mRNA primer issubstantially non-denatured; raising the temperature to a hightemperature, wherein the self-complementary stem of the hot start mRNAprimer is substantially denatured; hybridizing the target specificregion of the hot start mRNA primer to the target mRNA at the hightemperature; extending the hot start mRNA primer at the high temperatureto form a mRNA extension product; forming the mRNA primer extensionproduct and the micro RNA primer extension product in the same reactionmixture; pre-amplifying the mRNA primer extension product and the microRNA primer extension product in a pre-amplification PCR; performing afirst decoding PCR on the mRNA primer extension product and a seconddecoding PCR on the micro RNA primer extension product; and, quantifyingthe mRNA and the micro RNA from the small sample.

In some embodiments of the present teachings, a non hot-start primer canbe employed that nonetheless comprises a self-complementary stem and aloop. Such primers will normally have a single-stranded target specificregion that can hybridize to its corresponding target at a lowtemperature. Additional discussion of such primers can be found forexample in U.S. Non-Provisional applications to Chen et al., Ser. No.0/947,460 and Ser. No. 11/142,720. FIG. 1 depicts a scenario in which anon hot-start primer for a micro RNA target comprises aself-complementary stem, a loop, and single stranded target specificregion, is present in a reaction mixture along with a hot start mRNAprimer.

Generally, the length of the stem of the hot start primer can varyaccording to the context of the application. For example, when thetarget-specific region of the hot start primer is G:C rich, the lengthof the stem region can be shorter. Conversely, when the target-specificregion of the hot start primer is A:T rich, the length of the stemregion can be longer. Such procedures can be employed to adjust thelength of the stem to correspond with a desired Tm, given a particularreaction context at hand. In some embodiments, the length of the stem isbetween 6-12 nucleotide base pairs.

Generally, the length of the loop of the hot-start primer will bebetween 8-24 nucleotides in length. Generally, short loops can have thebeneficial effect of minimizing the likelihood of loop sequencedisplacing self-complementary stem duplex sequence at lower reactiontemperatures. It will be appreciated by one of ordinary skill in the artthat a variety of stem-loop configurations are available and withinroutine experimentation.

Illustrative molecular biology techniques of ready availability to oneof skill in the art can be found in Sambrook et al., Molecular Cloning,3rd Edition.

Certain Exemplary Kits

The instant teachings also provide kits designed to expedite performingcertain of the disclosed methods. Kits may serve to expedite theperformance of certain disclosed methods by assembling two or morecomponents required for carrying out the methods. In certainembodiments, kits contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits include instructions for performing one or more of the disclosedmethods. Preferably, the kit components are optimized to operate inconjunction with one another.

Thus, in some embodiments the present teachings provide a kit forforming a messenger RNA (mRNA) primer extension product and a micro RNA(miRNA) primer extension product in the same reaction mixturecomprising; a hot start mRNA primer, a micro RNA primer, and optionallya reverse transcribing enzyme, wherein the hot start mRNA primercomprises a blunt-ended self-complementary stem and a loop, wherein theblunt-ended self-complementary stem comprises a target specific regionand a quencher region, wherein the self-complementary stem isnon-denatured at a low temperature, wherein the low temperature is lessthan 27 C, and wherein the self-complementary stem is denatured at ahigh temperature, wherein the high temperature is between 35 C-60 C. Insome embodiments, the micro RNA primer comprises a self-complementarystem, a loop, and a single-stranded target specific region, wherein theself-complementary stem is non-denatured at the low temperature.

Although the disclosed teachings have been described with reference tovarious applications, methods, and kits, it will be appreciated thatvarious changes and modifications may be made without departing from theteachings herein. The foregoing examples are provided to betterillustrate the present teachings and are not intended to limit the scopeof the teachings herein. Certain aspects of the present teachings may befurther understood in light of the following claims.

1. A method of forming a messenger RNA (mRNA) primer extension productand a micro RNA primer extension product in the same reaction mixturecomprising; forming a reverse transcription reaction mixture comprisinga hot start mRNA primer, a micro RNA primer, a target mRNA, a targetmicro RNA, and a reverse transcribing enzyme, wherein the hot start mRNAprimer comprises a blunt-ended self-complementary stem, and a loop,wherein the blunt-ended self-complementary stem comprises a targetspecific region and a quencher region; hybridizing the micro RNA primerto the target micro RNA at a low temperature; extending the micro RNAprimer at the low temperature to form a micro RNA extension product,wherein the self-complementary stem of the hot start mRNA primer isnon-denatured; raising the temperature to a high temperature, whereinthe self-complementary stem of the hot start mRNA primer is denatured;hybridizing the target specific region of the hot start mRNA primer tothe target mRNA at the high temperature; extending the hot start mRNAprimer to form a mRNA extension product; and, forming the mRNA primerextension product and the micro RNA primer extension product in the samereaction mixture.
 2. The method according to claim 1 wherein the microRNA primer comprises a self-complementary stem, a loop, and asingle-stranded target specific region, wherein the self-complementarystem is substantially non-denatured at both the low temperature and thehigh temperature.
 3. The method according to claim 2 wherein theself-complementary stem of the micro RNA primer is 6-12 nucleotidebase-pairs in length and the single stranded target specific region is6-8 nucleotides in length.
 4. The method according to claim 1 whereinthe low temperature is 18 C-27 C.
 5. The method according to claim 1wherein the high temperature is 35 C-60 C.
 6. The method according toclaim 1 wherein blunt-ended self-complementary stem comprises between6-12 nucleotide base-pairs in length.
 7. A method of forming a firstprimer extension product and a second primer extension product in thesame reaction mixture comprising; forming a primer extension reactionmixture comprising a first primer, a hot start second primer, a firsttarget, a second target, and a primer extending enzyme, wherein the hotstart second primer comprises a blunt-ended self-complementary stem anda loop, wherein the blunt-ended self-complementary stem comprises atarget specific region and a quencher region; hybridizing the firstprimer to the first target at a low temperature; extending the firstprimer at the low temperature to form a first primer extension product,wherein the self-complementary stem of the hot start second primer isnon-denatured; raising the temperature to a high temperature, whereinthe self-complementary stem of the hot start second primer is denatured;hybridizing the target specific region of the hot start second primer tothe second target at the high temperature; extending the hot startsecond primer at the high temperature to form a second target extensionproduct; and, forming the first primer extension product and the secondprimer extension product in the same reaction mixture.
 8. A reactionmixture comprising a hot start mRNA primer, a micro RNA primer, a targetmRNA, a target micro RNA, and a reverse transcribing enzyme, wherein thehot start mRNA primer comprises a blunt-ended self-complementary stemand a loop, wherein the blunt-ended self-complementary stem comprises atarget specific region and a quencher region, wherein theself-complementary stem of the hot start mRNA primer is non-denatured ata low temperature, and wherein the self-complementary stem of the hotstart mRNA primer is denatured at a high temperature.
 9. The reactionmixture according to claim 8 wherein the micro RNA primer comprises aself-complementary stem, a loop, and a single-stranded target specificregion, wherein the self-complementary stem is substantiallynon-denatured at the low temperature.
 10. The reaction mixture accordingto claim 9 wherein the self-complementary stem of the micro RNA primeris 6-12 nucleotide base-pairs in length and the single-stranded targetspecific region is 6-8 nucleotides in length.
 11. The reaction mixtureaccording to claim 8 wherein the low temperature is 18 C-27 C.
 12. Themethod according to claim 8 wherein the high temperature is 35 C-60 C.13. The method according to claim 8 wherein blunt-endedself-complementary stem comprises between 6 and 12 nucleotide base pairsin length.
 14. A kit for forming a messenger RNA (mRNA) primer extensionproduct and a micro RNA (miRNA) primer extension product in the samereaction mixture comprising; a hot start mRNA primer, a micro RNAprimer, and optionally a reverse transcribing enzyme, wherein the hotstart mRNA primer comprises a blunt-ended self-complementary stem and aloop, wherein the blunt-ended self-complementary stem comprises a targetspecific region and a quencher region, wherein the self-complementarystem is non-denatured at a low temperature, wherein the low temperatureis less than 27 C, and wherein the self-complementary stem is denaturedat a high temperature, wherein the high temperature is between 35 C-60C.
 15. The kit according to claim 14 wherein the micro RNA primercomprises a self-complementary stem, a loop, and a single-strandedtarget specific region, wherein the self-complementary stem isnon-denatured at the low temperature.